生物大分子相互作用 Interaction between Biomacromolecules (interactome interactome)

Size: px

Start display at page:

Download "生物大分子相互作用 Interaction between Biomacromolecules (interactome interactome)"

规曾
5 years ago
Views:

1 二概论分子细胞生物学前沿进展与技术

2 生物大分子相互作用 Interaction between Biomacromolecules (interactome interactome)

3 The biomacromolecules in cell Protein DNA RNA Others

4 Why do we need to explore the interaction? By their friends, you shall know them!

5 The interaction of biomacromolecules is the fundamental basis of any cellular function RNA Protein Interaction DNA others

6 The focuses: Protein-protein interaction Protein-DNA interaction Protein-RNA interaction

7 The importance of protein-protein interaction Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and networks. Protein-protein interactions are involved in almost every aspects of biological processes. Protein-protein interactions provide potential drug targets.

8 The methods commonly used for studying protein-protein interaction Chemical crosslinking 化学交联 Pull-down 沉降实验 Co-immunoprecipitation (Co-IP) 免疫共沉淀 Yeast Two-hybrid (Y2H) 酵母双杂交 Fluorescence Resonance Energy Transfer (FRET) 荧光共振能量转移 Docking 嵌合计算 Biacore (Surface Plasmon Resonance, SPR) 表面等离子共振

9 Chemical Crosslinking Principle: physically coupling two proteins with a chemical crosslinker Advantage: in vivo Crosslinking reagents: 1. formaldehyde 2. photo-activated crosslinking reagents L-Photo-Leucine L-Photo-Methionine Subsequent analysis: SDS-PAGE, Western blotting, etc

10 Pull-down GST pull-down, His pull-down

11 Example: Detecting the interaction between FS and ANG using His pull-down experiment Western blot result of His pull-down How to further improve the experiment design?

12 Co-Immunoprecipitation (Western Blot or MS)

13 Example: BAG2 was identified by Co-IP as a CHIP-binding protein CHIP: carboxyl terminus of Hsp70- interacting protein 2005 JBC, 280:

14 Yeast Two-hybrid Principle: the modular design of eukaryotic transcription factors, which often have separate DNA-binding and transcriptional activation domains. Transcription factor: DB + AD DB: usually fuse to bait protein AD: prey protein or a cdna libray

15 Yeast Two-hybrid Basis of the Two-Hybrid System. (GIBCO BRL instruction manual)

16 Yeast Two-hybrid a gold standard for detecting interactions in vivo advantages: in vivo high-throughput screening disadvantages: yeast (cellular environment; protein modification) over-expressed protein not working with membrane proteins need to be confirmed by other methods

Development of Two-hybrid Technique Promega's CheckMate system: works in mammalian cells, more likely to have the proper folding and post-translational modifications split-ubiquitin system: good for

17 Development of Two-hybrid Technique Promega's CheckMate system: works in mammalian cells, more likely to have the proper folding and post-translational modifications split-ubiquitin system: good for membrane proteins ubiquitin: could be expressed as an N-terminal half (Nub) + a C-terminal half (Cub). The two halves retain their affinity for each other and spontaneously reassemble to form the so-called split-ubiquitin, but not if a point mutation is generated (NubG)

18 Fluorescence Resonance Energy Transfer (FRET) Fluorescence

19 GFP and other FPs (fluorescent proteins)

20 FRET: principle FRET is a distance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor)

FRET: basic requirements The donor emission spectrum must significantly overlap the excitation spectrum of the acceptor 供体发射波与受体激发波有重叠 The donor and

21 FRET: basic requirements The donor emission spectrum must significantly overlap the excitation spectrum of the acceptor 供体发射波与受体激发波有重叠 The donor and acceptor fluorophores must be in favorable mutual orientation 合适的空间取向 The distance between the donor and acceptor fluorophores must be less than 100 Å 距离 <100 Å

22 Detecting methods Monitor changes in donor fluorescence 检测供体荧光 Monitor changes in acceptor fluorescence 检测受体荧光 Simultaneously measure changes in both donor and acceptor fluorescence using spectral imaging 同时检测受体和供体荧光

23 Spectral imaging

24 Photobleaching

25 Photobleaching: an example

26 FRET: biosensor e.g. Prostate Specific Antigen (PSA): specifically recognize SSYYSG CFP G S G S G S S S Y Y S G G S G S G S YFP His6 CFP S S Y Y S G YFP His6

27 Docking Give the 3D structure of a protein target, compounds can be designed using bioinformatics methods to fit in a cavity, which is called "docking". Docking is an energy-based operation for exploring the binding modes of two interaction molecules. The treatment ends when a minimum of energy is obtained for the complex.

28 Goals of Docking To study ligand-protein, protein-protein and protein-dna interactions The influences of mutations on the interaction Drug screening

29 Softwares AutoDock Dock GOLD

30 surface plasmon resonance technology (SPR) 表面等离子共振技术 20 世纪 90 年代发展起来的一种基于 SPR 检测生物传感芯片 (biosensor chip) 上配位体与分析物作用的生物分子检测技术 SPR 利用金属膜 / 液面界面光的全反射引起的物理光学现象来分析分子相互作用

31 surface plasmon resonance technology (SPR) 表面等离子共振技术原理 : 偏振光在玻片 - 金膜界面全反射, 激发金膜中的自由电子产生表面等离子体当光波与表面等离子体的振频相等时, 二者发生谐振, 光能被吸收当入射光波长固定时, 反射光强度是入射角的函数反射光强度最低时所对应的入射角称为共振角实验中, 根据不同情况下共振角的位置变化, 来反映界面上液体的变化生物分子的相互作用将导致金属薄膜表面介电性质 ( 介电常数折射率 ) 发生改变

33 SPR: advantages & applications 优点 : 无需对样品进行标记实时监测灵敏度高快速用处 : 特异性哪些分子发生了相互作用? 浓度存在多少结合分子? 动力学相互作用的速率结合和解离的比例是多少? 亲和性相互作用的程度有多大? 协同作用是否存在任何异构效应? 相互作用模式结合模式与不同样品是否存在对应关系?

34 SPR: applications Drug screening Kinetics studies Cell signaling Multi-molecular complexes Enzyme reaction Ligand-receptor reaction Vaccine development Cancer reasearch Proteomics

35 SPR: further development 生物分子相互作用分析 - 质谱 (BIA-MAS): 是 SPR-BIA 技术与传统的蛋白鉴定技术 MALDI-TOF 有机结合而形成的一种新的研究手段 TRENDS in Biotechnology Vol.21 No.7 July 2003

36 Protein-protein Interaction Databases BIND (Biomolecular Interaction Network Database) DIP (Database of Interacting Proteins) IntAct HPRD (Human Protein Reference Database) MINT (Molecular INTeraction database) MIPS (Mammalian Protein Protein Interaction Database)

37 Protein-protein Interaction Databases 名称地址蛋白质数量 * PPI 数量 * 数据来源数据存储格式是否提供可视化网络分析特性 BIND 未知 >200,0 00 文献挖掘高通量 XML,ASN. 1, BioPAX,C SV,SIF,X ML 否 Ontoglyph DIP 19, ,168 文献挖掘 MIF,XIN 是 PPI 真实性评估 IntAc t ntact/site 56, ,93 1 文献挖掘 PSI-MI 是控制词汇, 预测 pulldown 实验 HPRD 25, ,581 文献挖掘 PSI-MI 否 PhosphoMot if Finder MINT a2.it/mint/ 28, ,80 8 文献挖掘 PSI-MI, Osprey 是 HomoMINT MIPS >900 >1,800 文献挖掘 PSI- MI,XML 否 PEDANT

38 The usage of PPI resources Protein-protein interactions: binary interactions, molecular complexes The network of interacting proteins Pathways (functional) Evolution of protein-protein interaction

39 The possible interaction network of angiogenin and its partners

40 Further Reading google: protein interaction; co-immunoprecipitation; CheckMate system; split-ubiquitin system

41 思考血管生成素 (angiongenin) 是一个血管生成和肿瘤相关蛋白质, 可以进入到靶细胞 ( 血管内皮细胞肿瘤细胞 ) 内, 并影响细胞行为请你设计一个实验, 筛选该蛋白的相互作用蛋白质

時間

104 學年度生醫系碩士班新生手冊 104 學年度生醫系碩士班新生座談會時間 :104 年 7 月 9 ( 星期四 ) 1:30 PM 地點 : 正心 222 程序 : 1. 新生第二階段報到 1:20~1:30pm 2. 師資課程等相關事項介紹 1:30~2:00 pm 3. 各老師領域專